Protein phosphorylation and dephosphorylation are known as important regulatory mechanisms which are used for signal transduction in various stages of the cellular functions. On the whole, cellular signals are mediated through phosphorylation and dephosphorylation, catalyzed by kinases and phosphatases, respectively. Due to their characteristic activities, particularly, protein phosphatases (PPases), which are responsible for dephosphorylation, are known to play pivotal roles in the in vivo modulation and regulation of fundamental cellular signaling mechanisms for metabolism, growth, proliferation and differentiation. Among these protein phosphatases are PTP1B, functioning to remove phosphate from tyrosine, Prl-3, LAR, CD45, Cdc25A, Cdc25B, Cdc25C, Yop, PP1, VHR and the like. Hereinafter, the results of the prior researches related to these PPases will be described.
1. PTP1B
PTP1B as the first identified intracellular protein tyrosine phosphatase, was isolated from the human placenta and found to have molecular weight of ˜50 KDa (Tonks et al., J. Biol. Chem. 1988, 263, 6722), and then successfully cloned (Charbonneau at al., Proc. Natl. Acad. Sci. USA 1989, 86, 5252; Chernoff et al., Proc. Natl. Acad. Sci. USA 1989, 87, 2735). PTP1B is highly expressed in various human cells. Particularly, PTP1B acts to inhibit the phosphorylation not only of insulin receptor (IR) but also of insulin receptor substrate (IRS-1) in the signal transduction pathway of insulin. It was demonstrated through the biochemical experiment conducted by Kennedy and Ramachandran, in which PTP1B knock-out mice were observed to exhibit insulin sensitivity and, when injected with insulin, to increase the phosphorylation of insulin receptors in hepatic and muscular cells (Science 1999, 283, 1544). Diabetes mellitus type II, a insulin-independent diabetes, is a metabolic disorder that is primarily characterized by insulin resistance of the organs (muscles, liver, lipocytes) where insulin disfunctions, although the pancreas normally secretes insulin. The dephosphorylation of the insulin receptor (IR) was found to be directly responsible for insulin resistance and be implicated in diabetes mellitus type II. Having potential activity to overcome insulin resistance and normalize the level of glucose and insulin in the blood without causing hypoglycemia, accordingly, PTP1B inhibitors, acting against the dephosphorylation of IR, have been extensively studied with the aim of developing therapeutic agents for diabetes mellitus type II.
For example, Wyeth-Ayerst and American Home Products disclosed 10 or more patents in which a broad spectrum of PTP1B inhibitors is described (U.S. Pat. No. 6,121,271, U.S. Pat. No. 6,110,963, U.S. Pat. No. 6,110,962, U.S. Pat. No. 6,103,708, U.S. Pat. No. 6,063,815, U.S. Pat. No. 6,057,316, U.S. Pat. No. 6,001,867, WO 9961436, WO 9961410, WO 9958522, WO 9958521, WO 9958520, WO 9958519, WO 9958518, WO 9958514, WO 9958511), and published many relevant articles (Wrobel, J. et al., J. Med Chem 1999, 42, 3199, Malamas, M. S. et al., J. Med. Chem. 2000, 43, 995; Malamas, M. S. et al., J. Med. Chem. 2000, 43, 1293; Wrobel, J. et al., Bioorg. & Med. Chem. Lett. 2000, 10, 1535). Through the above studies of PTP1B inhibitors, new compounds, including benzofuran and benzothiophene biophenyl, were reported to have IC50 values of tens of nM. A material which is also able to more effectively reduce the level of glucose in the blood than ciglitazone, which has been widely used as a therapeutic agent for diabetes mellitus, was found through in vivo experiments with mice. Recently, the development of the ertiprotafib, a kind of PTP1B inhibitor, represented by the following structural formulas, has been discontinued in phase II clinical testing.

Also, Abbott Company has continuously tried to develop PTP1B inhibitors on the basis of various chemical skeletons. (WO0264840, US 2002-077347, US 2002-072516, Diabetes 2002, 57(8), 2405). Prof. Zhang, Albert Einstein College of Medicine of Yeshiva University, suggested novel structures for PTP1B inhibitors by virtual screening, and also by molecular modeling using PTP1B crystalline structure (Zhang, Z. et al., J. Biol. chem. 2002, 277 (35), 31818. J. Med. Chem. 2000, 43, 146, Bioorg. & Med. Chem. Lett. 2000, 10, 457, Bioorg. & Med. Chem. Lett. 2000, 10, 923, Bioorg. & Med. Chem. Lett. 1998, 8, 2149, Bioorg. & Med. Chem. Lett. 1998, 8, 1799, Biochemisty 1999, 38, 3793).
Novo Nordisk/Ontogen Company introduced a low molecular weight oxalylaminoaryl acid derivative, which is neither derived from phosphatic acid nor from peptides (WO 9946237, 9946267, 0117516, Iversen, L. F. et al., J. Med. Chem. 2002, 45 (20), 4443; J. Biol. Chem. 2000, 275, 10300; Moller, N. P. H. et al., J. Biol. Chem. 2000, 275, 7101). According to their research, they emphasized that the synthetic PTP1B inhibitor has high selectivity for other protein tyrosine phosphatases.

Continuing to study PTP1B inhibitors, Merck Frosst Canada suggested α,α-difluoromethylenephosphonates structures (WO 0146206, 0146205, 0146204, 0146203, 0069889, 0017211, 0006712, Taylor, S. D. et al., Bioorg. & Med. Chem. Lett. 1998, 8, 345; Taylor, S. D. et al., Bioorg. Med. Chem. 1998, 6, 1457).
There are a lot of related articles published on the subject of PTP1B inhibitors (Lilijebris et al., Bioorg. & Med. Chem. Lett., 2002, 10, 1; Park et. al. Biochemistry 2002, 41, 9043; Cristopher et al., J. Med. Chem. 2002, 45 (18), 3946; Choi et al., Bioorg. & Med. Chem. Lett., 2002, 12 (15), 1941; Bleasdale, J. E. et al., Biochemisty 2001, 40, 5642; Umezawa, K. et al. Tetrahedron 2000, 56, 741; Taylor, S. C. et al., J. Chem. Soc. Perkin Trans 72000, 1271; Yokomatsu, T. et al., Bioorg. & Med. Chem. Lett. 1999, 9, 529).
Although there are such a large quantity of results as mentioned above, no materials have passed the complete clinical testing thus far. So there is an urgent need to find novel compounds which can be used in humans.
2. CD45
For cells to grow normally, highly elaborate signals are required. When the balance between them is broken and activation signals remain alone, cells grow uncontrollably, thus resulting in the occurrence of disorders. CD45 plays an essential role in terminating the signal transduction responsible for the uncontrollable growth of cells. CD45, a transmembrane PTPase (Protein Tyrosine Phosphatase), was known as an important role in signal transduction in T-cell or B-cells. In CD45 knock-out mice, Janus kinase (JAK) and STAT (signal transducer and activators of transcription) are observed to be activated by cytokines and interferon, demonstrating that CD45 interrupts the signal transduction of cytokines by inhibiting JAK.
In addition, CD45 negatively regulates interleukin-3-mediated cellular proliferation, erythropoietin-dependent hematopoiesis and antiviral responses. This indicates that CD45 suppresses the activity of the immune system of attacking foreign invaders, leading to the suppression of cancer cell proliferation and autoimmune diseases. Based on this finding, CD45 inhibitors can be available to prevent transplant rejection. In fact, extensive research into CN45 inhibitors has been conducted and the results thereof are disclosed in many patents and articles, issued to AstraZeneca Company (WO 0146125, 0145681, 0145680, R. A. Urbanek et al., J. Med. Chem. 2001, 44, 1777 and to others (JP2001114678, JP2001114689, WO 0128991, 0119830, 0119831, 0116097, 0128991). However, in spite of the extensive research results, no materials have succeeded in passing clinical testing, and thus there is an urgent need for a novel material for CD45 inhibitors.
3. LAR
It was hypothesized that LAR might be involved in the physiological modulation of insulin receptor signaling in intact cells (Hashimoto et al., J. Biol. Chem. 1992, 267, 13811). This conclusion was reached from data obtained by comparing the rate of dephosphorylation/inactivation of purified IR using PTP1B as well as the cytoplasmic domains of LAR. To examine whether the transmembrane PTPase LAR can modulate insulin receptor signaling in vivo, antisense inhibition was recently employed (Kulas et al., J. Biol. Chem. 1995, 270, 2435). In this test, LAR protein levels were specifically suppressed by approximately 60% in a rat hepatoma cell line. This suppression of the LAR protein level was paralleled by an approximately 150% increase in the insulin-dependent autophosphorylation of the insulin receptor.
However, only a modest 35% increase in insulin receptor tyrosine kinase (IRTK) activity was observed, whereas reduced LAR levels resulted in a 350% increase in insulin-dependent phosphatidylinositol 3-kinase (PI 3-kinase) activity. The authors speculated that LAR could specifically dephosphorylate tyrosine residues, which are critical for PI 3-kinase activation, either on the insulin receptor itself or on a downstream substrate.
Therefore, LAR inhibitors are expected to be useful in the treatment of obesity, impaired glucose tolerance, diabetes mellitus, hypertension, and partially ischemic diseases. In spite of the results from extensive research and studies, there are no compounds that have passed clinical tests thus far. Therefore, there is a need for a novel material applicable to human bodies for the treatment of such diseases.
4. Cdc25B
A dual specific phosphatase (defined as a subclass within the PTPase family able to hydrolyze phosphate from phosphor-tyrosine as well as from phosphor-serine/threonine) is responsible for the activation of cyclin dependent kinase (CDK) by hydrolyzing inhibitory phosphate from tyrosine and threonine residues of CDK, which is implicated in the cell division cycle. A high level of CDK induces the activation of the MPF (M phase promoting factor) to increase the mitotic activity in the M phase of cell cycle, resulting in cell proliferation.
Accordingly, dual specific phosphatase inhibitors interfere with cell division to thus prevent cell proliferation. Cdc25, a kind of dual specific phosphatase, has been reported to have three homologues, Cdc25 A, Cdc25B, and Cdc25C, in human cells. Among them, Cdc25B is inferred to play an important role in carcinogenesis because it is the most highly expressed in cancer cells. Because inducing M phase arrest, therefore, Cdc25B inhibitors can be targets for developing anticancer agents. Extensive and intensive research has been studied on the inhibitors and the results have been reported (Otani, T. et al., J. of Antibiotics 2000, 53, 337; Lazo, J. S. et al., Bioorg. Med. Chem. Lett. 2000, 8, 1451).
5. VHR
VHR, a dual specific phosphatase, extracellularly regulates extracellular signal receptor kinase 1 (ERK1) and ERK2, both belonging to a subclass of mitogen-activated protein kinase (MAPK), to mediate mitogenic signaling. Since VHR is involved in controlling cell cycles, its inhibitors, like Cdc25B inhibitors, can be available as anticancer agents (Osada, H. et al., FEBS Letters 1995, 372, 54).
6. Prl-3
Genetic level changes occurred in colon cancer thus far was the inactivation of tumor suppressors. However, these low-molecular weight materials are not suitable as targets for a novel anticancer drug. A recent report (Sana et al., Science 2001, 294, 1343) says that the novel phosphatase Prl-3 is commonly overexpressed in the metastasis of various colon cancer cells. The activity of Prl-3 is essential in the metastasis of colon cancer, therefore, effective Prl-3 inhibitors may be a drug target which can provide a new turning point for the therapy of colon cancer in the metastasis phase.
As elucidated above, overexpression or activity alteration of various protein phosphatases are reported to cause various diseases. Therefore, compounds having inhibitory activity against the protein phosphatases, if developed, could be useful in the prevention and treatment of specific diseases, such as diabetes, autoimmune diseases, and various cancers. Leading to the present invention, intensive and thorough research into protein phosphatase inhibitors, conducted by the present inventors, resulted in the finding that compounds derived from rhodanine can inhibit the activity of protein phosphatases.